The present invention relates to the field of molecular biology, human and non-human genetics, and metabolism and physiology. In particular, this invention provides a method for discriminating between two or more alleles that differ by only a single nucleotide. Also contemplated are specific markers to be included in melting curve analysis and melting curve genotyping experiments.
Various publications or patents are referred to throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein. Complete citations of scientific publications are set forth in the text or at the end of the specification.
Single nucleotide polymorphisms (SNPs) represent the most abundant type of sequence variation in the human genome and can and should be useful tools for many diverse applications, including delineating the genetic architecture of complex traits and diseases, pharmacogenetics, forensics, and evolutionary studies.
Historically, genetic studies have been predicated on identifying and employing genetic variation to address problems of biological significance. The third generation genetic map comprised of single nucleotide polymorphisms (SNPS) is being expeditiously developed (Wang, D. G., et al. (1998) Science 280:1077-1082). SNPs are the most abundant type of sequence variation in the human genome and are useful tools in many diverse applications including disease gene mapping, evolution, pharmacogenetics, and forensics. An impressive SNP resource already exists as nearly 300,000 have been deposited into publicly accessible databases, such as the National Cancer Institute database, and others. However, without parallel progress in SNP genotyping technology, their true power and inherent benefits will not come into fruition.
Novel genotyping methods amenable to high-throughput analysis should ideally be gel-free, robust, inexpensive, and simple to perform. To this end, these requirements have inspired the development of a variety of genotyping assays, including the oligonucleotide ligation assay xe2x80x9cOLAxe2x80x9d (Landegren, U., et al. (1988) Science 241: 1077-80); genetic bit analysis xe2x80x9cGBAxe2x80x9d (Nikiforov T. T., et al. (1994) Nucleic Acids Res. 11:4167-75); mass spectroscopy (Griffin, T. J., et al. (1999) Proc Natl Acad Sci. 25: 6301-6306), xe2x80x9cchipxe2x80x9d technology (Wang., et al, supra), TaqMan (Livak, K. J., et al. (1995) PCR Methods Appl. 4:357-62), and dynamic allele specific hybridization xe2x80x9cDASHxe2x80x9d (Howell, W. M., et al. (1999) Nat. Biotechnology 17:87-88). Although many SNP genotyping methods have been developed, no single technology has emerged as being clearly superior due to limitations such as cost, complexity, and accuracy. In particular, each of these methods frequently require repeat experimentation, a high level of skill in order to perform the assays, and reagents that are costly. More importantly, currently available methods require removing samples from the reaction plate at one or more stages, such as PCR purification and the generation of single stranded DNA templates.
Kinetic PCR is predicated upon monitoring the fluorescence of a diagnostic probe once per PCR cycle (Wittwer, C. T., et al. (1997) BioTechniques 22: 130-138). Recently, the principles underlying kinetic PCR have been applied to SNP genotyping methods (Germer, S. and Higuchi, R. (1999) Genome Res 9:72-78; Howell et al., supra; Livak, et al., supra). For example, the double stranded DNA specific dye SYBR Green I (Molecular Probes, Eugene Oreg.) has been used to analyze the melting curves of PCR products. These PCR product melting curves are characterized by a rapid loss of fluorescence as the temperature is raised through the samples melting temperature (Tm) (Ririe, K. M., et al. (1997) Analy. Biochem. 245: 145-160). Melting temperature is a function of solution buffer, product length, sequence composition, and GC content. Thus, it should be possible to distinguish DNA fragments that differ with respect to these parameters by melting curve analysis (MCA).
Two genotyping methods have been developed that rely on MCA. The first of these entails the use of allele specific PCR whereby one of the allele specific primers contains a GC tail (Germer, et al., supra). The allele specific PCR products are then subjected to MCA and different alleles are resolved based upon which allele specific primer led to amplification. While this represents an important advance in MCA applied to SNP genotyping, it is subject to the inherent limitations of allele-specific PCR, such as difficulty in reaction optimization. This difficulty in reaction optimization decreases the overall genotyping throughput and increases the effort required to develop new SNP markers.
Another method genotypes SNPs by analyzing the melting curves of short oligonucleotide probes hybridized to a region containing the SNP of interest. Two probes are used in these reactions, each one being complimentary to a particular allele at the SNP in question. Perfectly matched probes are more stable and have a higher melting temperature compared to mismatched probes. Hence, SNP genotypes are inferred according to the characteristic melting curves produced by annealing and melting either matched or mismatched oligonucleotide probes. Similar to the first method described above, this assay has several problems including an involved experimental strategy. Specifically, PCR must be performed with biotinylated primers to allow for attachment to streptavidin coated plates. In addition, the double strand DNA must be denatured to single strand DNA with an alkali solution, followed by hybridization of the probes and finally MCA.
In order for contemporary human genetics to realize its full potential, technological advances which allow high throughput, accurate, and low-cost genotyping must be developed. Current methods for SNP genotyping have several problems such as high cost and the requirement for multiple manipulations in the laboratory. Both of these result in limitations on genotyping throughput and require highly skilled technical support. Thus, there is a particular need for gel-free single nucleotide polymorphism (SNP) genotyping methods.
The present invention provides a method for the effective gel-free analysis of single nucleotide polymorphisms. The inventive method comprises three steps: (1) DNA amplification; (2) restriction enzyme digestion; and (3) melting curve analysis. In preferred embodiments, the amplification is PCR and the DNA analyzed is less than 120 base pairs in length. Preferably, amplification, restriction enzyme digestion, and MCA are conducted in the same reaction tube(s). In preferred embodiments, the reaction and analysis time is less than 20 minutes. In highly preferred embodiments, the analysis time is less than 5 minutes.
One embodiment of the inventive method comprises the steps of amplifying at least one DNA segment of pre-determined size wherein the SNP, if present, is located, the amplification further comprising introducing a selected restriction endonuclease recognition site into the segment if the segment does not contain the selected restriction endonuclease recognition site; digesting the amplification product with a restriction enzyme to produce a digested DNA product; and analyzing the melting curve of the digested DNA product by Melting Curve Analysis.
DNA amplification is conducted using standard techniques. In preferred embodiments, amplification method is PCR and comprises the steps of initial denaturation, denaturation, annealing, polymerization, and final extension. Preferably, denaturation is conducted at between 90-95xc2x0 C. for 10-30 seconds. In highly preferred embodiments, denaturation is conducted at 95xc2x0 C. for 30 seconds. Preferably, annealing is conducted at 45xc2x0 C.-65xc2x0 C. for 10-60 seconds. In highly preferred embodiments, annealing is conducted at 55xc2x0 C. for 30 seconds. Preferably, extension is conducted at 72xc2x0 C. for 10-90 seconds, and the final extension is conducted at 72xc2x0 C. for 5 minutes. In preferred embodiments, the reaction mixture comprises genomic DNA, MgCl2, 10xc3x97PCR buffer, 0.1 mM dNTPs, 0.04 uM of each primer, and 2.5 units of heat stable polymerase. In highly preferred embodiments, the heat stable polymerase is Taq polymerase. Optionally, the reaction mixture may contain SYBR Green I and Formamide or DMSO.
A critical requirement of the McSNP assay is the presence of a restriction site in the DNA generated by the SNP in one allele and not the other. In one embodiment, the restriction site is naturally occurring in one allele. In an alternative embodiment, the restriction site may be generated synthetically. The synthetic restriction site on one of the SNP alleles can be introduced by designing one of the PCR primers with a mismatch near the 3xe2x80x2 end. In preferred embodiments, restriction enzyme reactions are comprised of PCR product, restriction enzyme, 1xc3x97reaction buffer specific for the restriction enzyme, and optionally, 1xc3x97Bovine Serum Albumin.
Preferably, melting curve analysis is performed measuring or detecting the decrease of double stranded DNA or increase of single stranded DNA. In one embodiment, double-strand DNA is detected using a dye or probe. In a preferred embodiment, the dye is a fluorescent dye. In a highly preferred embodiment, the dye is SYBR Green I. In an alternative embodiment, single-strand DNA is measured using a dye or probe. In a preferred embodiment, the dye is a fluorescent dye. In a highly preferred embodiment, the dye is SYBR Green II.
In preferred embodiments, a destabilizing agent may be added to the Melting Curve Reaction to destabilize double strand DNA and lower the Tm. In highly preferred embodiments, the destabilizing agent is DMSO and/or formamide. Optionally, automatic scoring software may be used to classify the genotypes after McSNP.
Also provided is a method for the solid phase analysis of McSNP experiments which comprises the steps of multiplexing PCR, restriction enzyme digestion, and analysis steps performed on a solid phase. In one preferred embodiment, the solid phase analysis is a Primer Directed Protocol based method. In another preferred embodiment, the solid phase analysis is Whole Product Hybridization based method.
Another aspect of the invention features specific DNA fragments to be included in melting curve analysis and melting curve SNP genotyping experiments. Methods of making melting point markers are also included in the present invention and comprise selecting a DNA sequence is that has the appropriate length and sequence for the melting point required; synthesizing the two strands of the sequence; mixing equimolar amounts of the sequences together; heating the mixture of sequences to 90xc2x0 C. for approximately one minute; and allowing the mixture to cool in order to anneal the oligos together. In an alternative embodiment, the mixture of sequences are treated with a denaturating chemical gradient instead of heating. Acceptable denaturating chemicals include DMSO or formamide. Optionally, the oligos can be further labeled for fluorescence resonance energy transfer using a fluorescent dye that does not overlap in its emission spectrum with the double stranded DNA specific fluorescent dye. The annealed melting point marker is added to the restriction digested PCR product at the same time as the double strand DNA specific fluorescent dye and formamide.